Wickwire B M, Wagner C, Broquist H P
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
J Biol Chem. 1990 Sep 5;265(25):14748-53.
The fungal parasite Rhizoctonia leguminicola produces two indolizidine alkaloids, slaframine and swainsonine, of physiological interest. These alkaloids are biosynthesized from pipecolic acid which in turn is derived from L-lysine in this fungus as shown in the accompanying paper (Wickwire, B.M., Harris, C.M., Harris, T.M., and Broquist, H.P. (1989) J. Biol. Chem. 265, 14742-14747): L-lysine----saccharopine----delta 1----piperideine-6- carboxylate----pipecolate. This paper concerns the discovery, purification, and properties of a flavoenzyme, termed saccharopine oxidase, which carries out the oxidative cleavage of saccharopine as follows: Saccharopine + O2----delta 1-piperidine-6-carboxylate + glutamate + H2O2 The enzyme was purified 2,000-fold to homogeneity (polyacrylamide gel electrophoresis) in 14% yield from R. leguminicola mycelia, and had a native molecular mass of about 45,000 daltons by gel filtration (fast protein liquid chromatography Superose). Evidence for the presence of a flavin in the enzyme was drawn from these considerations: (a) the enzyme, while oxidatively cleaving saccharopine, concomitantly reduces 2,6-dichlorophenolindophenol; (b) the purified enzyme has a fluorescence spectrum typical of flavins; and (c) the enzyme requires oxygen and produces hydrogen peroxide. Good correlation was shown with purified saccharopine oxidase between disappearance of saccharopine with the concomitant appearance of delta 1-piperideine-6-carboxylate plus glutamate. The enzyme has a pH optimum about 6 and a Km for saccharopine of 0.128 mM. The enzyme apparently exists in R. leguminicola to shunt saccharopine, a major lysine metabolite, into a secondary pathway of lysine metabolism leading to pipecolate and subsequently to slaframine and swainsonine.
真菌寄生虫豆科丝核菌产生两种具有生理活性的吲哚里西啶生物碱，即司拉明和苦马豆素。这些生物碱由哌啶酸生物合成，而哌啶酸又由该真菌中的L-赖氨酸衍生而来，如随附论文所示（Wickwire, B.M., Harris, C.M., Harris, T.M., and Broquist, H.P. (1989) J. Biol. Chem. 265, 14742 - 14747）：L-赖氨酸→酵母氨酸→δ¹→哌啶-6-羧酸盐→哌啶酸。本文涉及一种黄素酶（称为酵母氨酸氧化酶）的发现、纯化及其性质，该酶催化酵母氨酸的氧化裂解反应如下：酵母氨酸 + O₂→δ¹-哌啶-6-羧酸盐 + 谷氨酸 + H₂O₂。该酶从豆科丝核菌菌丝体中纯化至同质状态（聚丙烯酰胺凝胶电泳），纯化倍数达2000倍，产率为14%，通过凝胶过滤（快速蛋白质液相色谱Superose）测定其天然分子量约为45,000道尔顿。从以下几方面考虑得出该酶中存在黄素的证据：(a) 该酶在氧化裂解酵母氨酸的同时，能使2,6-二氯酚靛酚还原；(b) 纯化后的酶具有典型的黄素荧光光谱；(c) 该酶需要氧气并产生过氧化氢。纯化的酵母氨酸氧化酶催化反应中酵母氨酸的消失与δ¹-哌啶-6-羧酸盐和谷氨酸的同时出现具有良好的相关性。该酶的最适pH约为6，对酵母氨酸的Km值为0.128 mM。该酶显然存在于豆科丝核菌中，将酵母氨酸（一种主要的赖氨酸代谢产物）分流到赖氨酸代谢的次要途径，进而生成哌啶酸，随后生成司拉明和苦马豆素。
